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Long non-coding RNAs (lncRNAs) have emerged as key regulators of cellular senescence by transcriptionally and post-transcriptionally modulating the expression of many important genes involved in senescence-associated pathways and processes. Among the different lncRNAs associated to senescence,Senescence Associated Long Non-coding RNA(SALNR) was found to be down-regulated in different cellular models of senescence. Since its release in 2015,SALNRhas not been annotated in any database or public repository, and no other experimental data have been published. TheSALNRsequence is located on the long arm of chromosome 10, at band 10q23.33, and it overlaps the 3’ end of theHELLSgene. This investigation helped to unravel the mystery of the existence ofSALNRby analyzing publicly available short- and long-read RNA sequencing data sets and RT-PCR analysis in human tissues and cell lines. Additionally, the expression ofHELLShas been studied in cellular models of replicative senescence, bothin silicoandin vitro. Our findings, while strongly questioning the actual existence ofSALNRas an independent transcript, support the expression of a predictedHELLSisoform entirely covering theSALNRgenomic region. Furthermore, we observed a strong down-regulation ofHELLSin senescent cells versus proliferating cells, supporting its role in the senescence and aging process.Graphical abstract

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin growth factor family, well known for its role in the homeostasis of the cardiovascular system. Recently, the human BDNF Val66Met single nucleotide polymorphism has been associated with the increased propensity for arterial thrombosis related to acute myocardial infarction (AMI). Using cardiac magnetic resonance imaging and immunohistochemistry analyses, we showed that homozygous mice carrying the human BDNF Val66Met polymorphism (BDNFMet/Met) undergoing left anterior descending (LAD) coronary artery ligation display an adverse cardiac remodeling compared to wild-type (BDNFVal/Val). Interestingly, we observed a persistent presence of pro-inflammatory M1-like macrophages and a reduced accumulation of reparative-like phenotype macrophages (M2-like) in the infarcted heart of mutant mice. Further qPCR analyses showed that BDNFMet/Met peritoneal macrophages are more pro-inflammatory and have a higher migratory ability compared to BDNFVal/Val ones. Finally, macrophages differentiated from circulating monocytes isolated from BDNFMet/Met patients with coronary heart disease displayed the same pro-inflammatory characteristics of the murine ones. In conclusion, the BDNF Val66Met polymorphism predisposes to adverse cardiac remodeling after myocardial infarction in a mouse model and affects macrophage phenotype in both humans and mice. These results provide a new cellular mechanism by which this human BDNF genetic variant could influence cardiovascular disease.

The identification of patient characteristics associated with SAMS may help to better understand and predict this unwanted effect of statins. The clinical data reported for patients with SAMS allows the identification of predisposing factors that can be divided into both endogenous (demographics, medical history) and exogenous (diet or lifestyle and concurrent medications) factors. Attention to the potential for muscle problems with statin therapy, in particular when they are given in combination with other drugs, in the presence of particular lifestyle conditions or dietary components should help reducing the risk of muscle events.

The use of warfarin, the most commonly prescribed oral anticoagulant, is being questioned by clinicians worldwide due to warfarin several limitations (a limited therapeutic window and significant variability in dose-response among individuals, in addition to a potential for drug-drug interactions). Therefore, the need for non-vitamin K antagonist oral anticoagulants (NOACs) with a rapid onset of antithrombotic effects and a predictable pharmacokinetic (PK) and pharmacodynamic (PD) profile led to the approval of five new drugs: the direct factor Xa (F-Xa) inhibitors rivaroxaban, apixaban, edoxaban and betrixaban (newly approved by FDA) and the direct thrombin (factor-IIa) inhibitor dabigatran etexilate. The advantages of NOACs over warfarin are a fixed-dosage, the absence of the need for drug monitoring for changes in anti-coagulation and fewer clinically significant PK and PD drug-drug interactions. NOACs exposure will likely be increased by the administration of strong P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4-inhibitors and may increase the risk of bleeds. On the contrary, P-gp inducers could significantly decrease the NOACs plasma concentration with an associated reduction in their anticoagulant effects. This manuscript gives an overview of NOACs PK profiles and their drug-drug interactions potential. This is meant to be of help to physicians in choosing the best therapeutic approach for their patients.

Background and aims Cholesterol-loaded smooth muscle cells (SMCs) modify their phenotypic behavior becoming foam cells. To characterize the role of ABCA1 and HDL3 in this process, we evaluated HDL3 effects on cholesterol-induced phenotypic changes in SMCs expressing or not ABCA1. Methods SMCs, isolated from the aortae of wild-type (WT) and Abca1 knock-out (KO) mice, were cholesterol-loaded using a “water-soluble cholesterol’’. Results Cholesterol loading downregulates the expression of Acta2 and calponin (SMC markers), and increases the expression of Mac-2, CD11b and MHCII (inflammation-related genes and surface antigens) and Abca1, Abcg1. HDL3 normalizes SMC marker expression and reduces the expression of inflammation-related genes/proteins in WT cells, an effect not observed with free apoA-I. The effect of HDL3 is almost lost in Abca1 KO cells, as well as when Abca1 is silenced in WT SMC. HDL3 does not differently affect cholesterol downloading in WT or KO cells and stimulates phospholipids removal in WT cells. Similarly, the expression of myocardin and its modulators, such as miR-143/145, is reduced by cholesterol loading in WT and Abca1 KO SMCs; HDL3 normalizes their levels in WT cells but not in KO cells. On the contrary, cholesterol loading induces Klf4 expression while HDL3 restores Klf4 to basal levels in WT cells, but again this effect is not observed in KO cells. Conclusions Our results indicate that HDL3, by interacting with ABCA1, modulates the miR143/145-myocardin axis and prevents the cholesterol-induced gene expression modification in SMCs regardless of its cholesterol unloading capacity.

Abnormal glycolytic metabolism contributes to angiogenic sprouting involved in atherogenesis. We investigated the potential anti-angiogenic properties of specific 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) inhibitors in endothelial cells (ECs). ECs were treated with PFKFB3 inhibitors (named PA-1 and PA-2) and their effects on metabolic and functional characteristics of ECs were investigated. The anti-glycolytic compound 3-(pyridinyl)− 1-(4-pyridinyl)− 2-propen-1-one (3PO) was used as reference compound. PFKFB3 expression and activity (IC50 about 3–21 nM) was inhibited upon treatment with both compounds. Glucose uptake and lactate export were measured using commercial assays and showed a partial reduction up to 40%. PFKFB3 inhibition increased intracellular lactate accumulation, and reduced expression of monocarboxylate transporters-1 (MCT1) and MCT4. Furthermore, endothelial cell migration and proliferation assays demonstrated significant reduction upon treatment with both compounds. Matrix- metalloproteinase (MMP) activity, measured by gelatin zymography, and expression was significantly reduced (up to 25%). In addition, PA compounds downregulated the expression of VCAM-1, VE-cadherin, VEGFa, VEGFR2, TGF-β, and IL-1β, in inflamed ECs. Finally, PA-1 and PA-2 treatment impaired the formation of angiogenic sprouts measured by both morphogenesis and spheroid-based angiogenesis assays. Our data demonstrate that the anti-glycolytic PA compounds may affect several steps involved in angiogenesis. Targeting the key glycolytic enzyme PFKFB3 might represent an attractive therapeutic strategy to improve the efficacy of cancer treatments, or to be applied in other pathologies where angiogenesis is a detrimental factor.

Aim: Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of the glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compounds with a potential to efficiently modulate (inhibit) angiogenesis. Methods: Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. Moreover, gene expression of important markers related to angiogenesis were measured at mRNA level by real-time PCR. Results: PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2 suppressed capillary tube formation, 3PO did not have any effect. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds. Conclusions: These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeutic approach to promote plaque stability.

Aim: Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of theglycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compoundswith a potential to efficiently modulate (inhibit) angiogenesis. Methods: Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. Moreover, gene expression of important markers related to angiogenesis were measured at mRNA levelby real-time PCR. Results: PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2suppressedcapillary tube formation, 3PO did not have any effect. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds. Conclusions: These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeuticapproach to promote plaque stability.

After more than a decade of intense investigation, Pro-protein Convertase Subtilisin-Kexin type 9 (PCSK9) remains a hot topic of research both at experimental and clinical level. Interestingly PCSK9 is expressed in different tissues suggesting the existence of additional function(s) beyond the modulation of the Low-Density Lipoprotein (LDL) receptor in the liver. Emerging data suggest that PCSK9 might play a role in the modulation of triglyceride-rich lipoprotein (TGRL) metabolism, mainly Very Low-Density Lipoproteins (VLDL) and their remnants. In vitro, PCSK9 affects TGRLs production by intestinal cells as well as the catabolism of LDL receptor homologous and non-homologous targets such as VLDL receptor, CD36 and ApoE2R. However, the in vivo relevance of these findings is still debated. This review aims at critically discussing the role of PCSK9 on TGRLs metabolism with a major focus on the impact of its genetic and pharmacological modulation on circulating lipids and lipoproteins beyond LDL.

Cholesterol-loaded smooth muscle cells (SMCs) modify their phenotypic behavior becoming foam cells. To characterize the role of ABCA1 and HDLSMCs, isolated from the aortae of wild-type (WT) and Abca1 knock-out (KO) mice, were cholesterol-loaded using a "water-soluble cholesterol''.Cholesterol loading downregulates the expression of Acta2 and calponin (SMC markers), and increases the expression of Mac-2, CD11b and MHCII (inflammation-related genes and surface antigens) and Abca1, Abcg1. HDLOur results indicate that HDL

Circulating monocytes adhere to the endothelium and migrate into the intima contributing to atherosclerotic plaque growth. Cigarette smoke is a risk factor for atherosclerosis, but it is not completely known how it affects monocyte behavior in atherogenesis.We studied the effects of cigarette smoke condensate (CSC) on human monocytes (HM) chemotaxis and transmigration through an endothelial cell (EC) monolayer.Pre-treatment with CSC caused a decrease in HM chemotaxis and transmigration (-55% and -18% vs control, p0.05, respectively), paralleled by a reduced expression of Rac 1 GTPase. On the contrary, direct exposure of both HM and EC to CSC increased (+23% vs control, p0.05) HM transmigration, paralleled by a strong stimulation of VCAM1 and ICAM1 expression by ECs, and by a slight increase in monocyte integrin expression. An enhancement of monocyte transmigration was obtained after the exposure of both HM and EC to medium conditioned by HM previously incubated with CSC (+265% vs control, p0.001). CSC showed a stimulatory effect on the expression by HM of TLR4, MCP1, IL8, IL1beta, and TNFalfa, which was ablated by pre treatment with PDTC. Incubation with neutralizing antibodies against both MCP1 or IL8 completely abolished the CSC-conditioned medium induced HM transmigration.CSC induces HM to release chemotactic factor(s), which amplify the recruitment and transmigration of inflammatory cells through EC, but CSC may also reduce HM migratory capacity. Therefore, exposure to CSC affects monocyte behavior and interaction with the endothelium, thus potentially facilitating and/or further aggravating the atherogenic process.

This work aims to preliminarily evaluate the reliability of regenerated keratins (RKs) in the design of microparticulate drug delivery systems by studying their processability and cytotoxicity. RKs were extracted by sulfitolysis from wool waste. A 4.5% w/w RK solution was spray-dried, and microparticles were sterilized by steam vapor under pressure. Scanning electron microscope, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Fourier transform infrared spectroscopy were used to characterize RKs and microparticles thereof. The in vitro cytotoxicity was determined by assessing the release of lactate dehydrogenase in the human monocytic cell line Tamm–Horsfall glycoprotein-1. RK-based microparticles with a narrow and unimodal particle size distribution (~6 µm) were obtained. They had a raisin-like structure with a smooth surface. Both microparticle morphology and RK molecular weight were well-preserved after sterilization. The curve fitting of the amide I bands showed that RK in the microparticles was prevalently present in the disordered/α-helix secondary structures which made the protein soluble in water. To promote crystallization in the β-sheet secondary structure and, therefore, water insolubility, RK-based microparticles were immersed in an aqueous solution of acetic acid at pH 3.5 overnight. RK did not induce any appreciable cellular cytotoxicity at any of the concentrations (from 1 up to 1000 µg sterile microparticles in 1 ml cell culture medium) or time-points (24–72 h) tested. These preliminary data suggest the feasibility of producing RK biocompatible microparticles using waste wool as starting material. Copyright © 2013 John Wiley & Sons, Ltd.

Cells of the artery wall may accumulate free cholesterol and cholesteryl esters becoming foam cells. Up to 50% of foam cells in human lesions originates from smooth muscle cells (SMCs). Arterial SMCs express the ATP binding cassette (ABC) transporter ABCA1 and, upon cholesterol loading, express macrophage markers and a phagocytic activity. To characterize the role of ABCA1 and HDL3 in this transdifferentiation process, we evaluated the phenotypic changes in SMCs isolated from wild type (WT) and ABCA1 knock out (KO) mice and how HDL3 affects these changes. Cholesterol loading causes the downregulation of the expression of SMC markers including ACTA2, alpha-tropomyosin and myosin heavy chain and increases the expression of macrophage-related genes such as CD68, Mac-2, SRB1, MMPs, ABCG1 and ABCA1. HDL3 treatment in WT cells is able to normalize the expression of ACTA2, while the expression of macrophage-related genes is reduced. On the contrary, the preventive effect of HDL3 is completely lost in ABCA1 KO cells. Interestingly, the presence of HDL3 does not differently affect neutral lipid accumulation in WT or ABCA1 KO cells but stimulates phospholipids removal only in WT cells. ApoAI addition does not reverse the phenotypic changes induced by cholesterol not only in KO but also in WT cells. Moreover, cholesterol loading reduces the expression of myocardin, the master SMC specific-transcriptional coactivator involved in SMC differentiation, by up to 55% (p

Statin monotherapy is generally well tolerated, with a low frequency of adverse events. The most important adverse effects associated with statins are myopathy and an asymptomatic increase in hepatic transaminases, both of which occur infrequently. Because statins are prescribed on a long-term basis, their possible interactions with other drugs deserve particular attention, as many patients will typically receive pharmacological therapy for concomitant conditions during the course of statin treatment.This review summarizes the pharmacokinetic properties of statins and emphasizes their clinically relevant drug interactions and related adverse reactions.Avoiding drug-drug interactions and consequent adverse drug reactions is essential in order to optimize compliance, and thus improve the treatment of patients at high cardiovascular risk. The different pharmacokinetic profiles among statins should be carefully considered, in order to understand the possible spectrum of drug interactions. The growing trend toward earlier statin treatment for the prevention of cardiovascular disease means that physicians must anticipate future polypharmacy when their patients require additional medications for comorbid conditions.

Objective To investigate whether the morphological and functional changes typical of cell immobilization induced by free cholesterol (FC) accumulation in macrophages is related to the activity of the ATP-binding cassette transporter (ABCA1). Methods and results FC loading induced actin rearrangement with ruffling and cell spreading in macrophages that normally express ABCA1, but to a significant lesser extent in ABCA1-KO mouse peritoneal macrophages (MPMs) and in normal cells upon pharmacological inhibition of ABCA1 with probucol. In ABCA1-KO MPMs and in probucol-treated J774 cell migration was inhibited to a lower extent by FC as compared to control cells. Similar results were found in stably ABCA1 knocked down J774 (ABCA1-KD-J774) obtained by RNA interference. FC accessible to cholesterol oxidase, a measure of plasma membrane FC content, was significantly higher in FC-loaded WT MPMs and control J774 than in FC-loaded ABCA1-KO MPMs, ABCA1-KD-J774 or probucol-treated J774. In parallel plasma membrane total phospholids and sphingomyelin increased after cholesterol loading in control J774 but not in ABCA1-KD-J774. In addition, apoA-I, that removes FC from ABCA1 specific pool, partially restored chemotactic response in FC-loaded control J774. No effect was observed with HDL 2 that does not interact with ABCA1. Finally, FC-induced Rac activation was more efficient in control J774 than in ABCA1-KD-J774. and was prevented by probucol and apoA-I in control J774. Conclusion In macrophages ABCA1 activity mediates FC ability to alter plasma membrane organization, to inhibit cell migration, and to activate a Rac-mediated signaling pathway.

Introduction: Hyperparathyroidism has been associated with essential hypertension. In rats, volume expansion, the deoxycorticosterone (DOC)-salt hypertension model and primary aldosteronism cause a reduction in plasma calcium levels and secondary hyperparathyroidism. In these rats, urinary calcium decreases with thiazides and/or aldosterone antagonists, while furosemide gives the opposite effect. Also, licorice causes an increase in parathyroid hormone and urinary calcium excretion.

Monocytes/macrophages recruited into the arterial wall during atherogenesis are crucial in the initiation and progression of atherosclerosis and play a fundamental role in the destabilization process that is the main causal event of acute coronary syndromes. In the present study, we investigated the effect of the mammalian target of rapamycin inhibitor everolimus on macrophage accumulation within carotid lesions elicited by perivascular collar placement in cholesterol-fed rabbits. Everolimus (1.5 mg/kg given 1 day before collaring followed by 1 mg/kg/day for 14 days, administered by oral gavage) markedly decreased lesion macrophage content as compared with vehicle control (-65%; p < 0.01). This effect was associated with a reduction in intimal thickening and occurred in the absence of changes in plasma cholesterol concentrations. To gain insights on the potential mechanism(s) underlying this effect, we investigated the influence of everolimus on chemoattractant-induced migration of human monocytes in vitro. Pretreatment with therapeutic concentrations of everolimus (10 nM) significantly lowered monocyte chemotaxis in response to various chemotactic factors (i.e., monocyte chemoattractant protein-1/CCL2, fractalkine/CX3CL1, interleukin-8/CXCL8, complement fragment 5a, or N-formyl-Met-Leu-Phe) without inducing monocyte cell death. These results suggest that everolimus may favorably influence the atherosclerotic process by affecting the recruitment of monocytes into early lesions.

Metalloproteinases (MMPs) are zinc-dependent endopeptidases responsible for the hydrolysis of various component of extracellular matrix such as gelatin and collagen. MMPs, namely MMP-2 and MMP-9 correlate with cardiovascular events in patients. We sought to determine whether supplementation with polyphenol-rich Cynara cardunculus (wild artichoke, traditional component of the Mediterranean diet) modulates MMP-9 expression and activity in cell cultures. A fully characterized C. cardunculus extract was able to inhibit, in a dose-dependent manner, the gelatinolytic activity of secreted MMP-9 and both secretion and human MMP-9 promoter-driven transcription. Analysis by HPLC of the Cynara extract identified polyphenols such as luteolin, apigenin, and caffeic acid, among others. However, testing a mix of the individual components suggested that the inhibitory effects of C. cardunculus are due to minor constituent fraction(s) as a whole. In promoting the health benefits of the Mediterranean diet, the role of wild plants as important meal components deserves further reappraisal.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that regulate both integrity and composition of the extracellular matrix (ECM). Excessive ECM breakdown by MMPs is implicated in many physiological and pathological conditions, such as atherosclerosis. Activated macrophages, especially in the atherosclerotic lesion, are a major source of reactive oxygen species (ROS). Antioxidants protect against ROS-induced MMPs activation and inhibit gelatinolytic activity. We sought to determine whether the antioxidants glutathione (GSH), N-acetylcysteine (NAC), or lipoic acid (LA) affect gelatinase production and secretion. The results show that thiol compounds affect MMPs expression and activity in different ways. MMP-2 activity is directly inhibited by NAC and GSH, while LA is ineffective. On the contrary, MMP-9 expression is inhibited by LA at a pretrascriptional level, and MMP-9 activity is stimulated by GSH through a direct interaction with the gelatinase itself. Although all thiols, these compounds have different properties and different cellular uptakes and metabolic characteristics, and this could explain, at least in part, their differential effects on MMPs.

Cigarette smoke induces endothelial cell (EC) dysfunction. Circulating monocytes (HM) adhere to a damaged endothelium and migrate into the intima, differentiate to macrophages and contribute to atherosclerotic plaque growth. We demonstrated that HM exposed to cigarette smoke condensate (CSC) increase the transmigration of HM through EC. Thus, we hypothesized that CSC may affect HM/EC interaction. We analyzed the effects on EC of CSC (30 μg/ml) alone and/or of medium conditioned by HM incubated without (CM) or with CSC (CM/CSC). CSC, CM or CM/CSC did not affect EC vitality (MTT assay). CM and CM/CSC increased the expression (measured by RT-PCR) of adhesion molecule ICAM1 (36-fold and 90-fold, p

The synthesis of trifluoromethyl (Tfm) analogs of known nanomolar matrix metalloproteinases (MMPs) inhibitors has been performed. The synthetic protocol is based on a moderately stereoselective aldol reaction of trifluoropyruvate with an N-acyl-oxazolidin-2-thione for the construction of the core α-Tfm-malic unit. Both the diastereomeric forms of the target α-Tfm-malic hydroxamates showed micromolar inhibitory potency toward MMP-2 and 9, according to zymographic tests, with a substantial drop with respect to the parent unfluorinated compounds. We also report some molecular modeling results, which provide a rationale for the experimental findings.

Catechins are able to modulate the gelatinolytic activity of matrix metalloproteinase-9 (MMP-9) by reducing its release from macrophages. Gallocatechins decrease MMP-9 secretion by lowering MMP-9 promoter activity and mRNA levels. The effect appears to be dependent on some structural and stereochemical requirements. In this study, the relationship between chemical structure and activity was studied by testing the effect of analogues of (+/-)-gallocatechin-3-gallate (+/-)-GCG, selectively deprived of hydroxyl groups, on MMP-9 activity, transcription, and secretion. Our results indicate that (+/-)-GCG and (+/-)-catechin-3-gallate are characterized by a substitution pattern compatible with direct inhibition of MMP-9 activity. Conversely, when transcription was the target, (+/-)-trans-3-flavanol-3-benzoate, lacking all the hydroxyl groups, was the most effective both in lowering MMP-9 promoter activity and consequently protein secretion, and in inhibiting nuclear-factor-kappaB-driven transcription. Our results suggest that the structural requirements for enzyme inhibition are different from those necessary for targeting gene expression.

The racemic α-trifluoromethyl-α-amino-β-sulfone hydroxamates 1 were synthesized by means of a nucleophilic addition of sulfur-stabilized carbanions to a N -Cbz imine of trifluoropyruvate ( 4 ). The free amino derivative 1a was the most potent inhibitor of both MMP-3 (stromelysin-1) and MMP-9 (gelatinase-B), showing an IC 50 = 14 nM and 1 nM, respectively, and excellent selectivity versus MMP-1 (>5000-fold difference in inhibitory capacity). The N -Me derivative 1b was the most selective for MMP-3 with respect to MMP-9 (62-fold difference).

Clinical trials have firmly established that 3-hydroxy-3-methylglutaryl-coenzyme-A reductase inhibitors (statins) can induce regression of vascular atherosclerosis as well as reduction of cardiovascular-related morbidity and death in patients with and without coronary artery disease. These beneficial effects of statins are usually assumed to result from their ability to reduce cholesterol synthesis. However, because mevalonic acid is the precursor not only of cholesterol but also of many nonsteroidal isoprenoid compounds, inhibition of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase may result in pleiotropic effects. Indeed, statins can interfere with major events involved in the formation and the evolution of atherosclerotic lesions, such as arterial myocyte migration and proliferation and cholesterol accumulation, independent of their hypolipidemic properties. The aim of this article is to focus on clinical and experimental data that show that statins possess effects beyond cholesterol lowering, particularly on arterial smooth muscle cell proliferation. The contribution of these direct vascular effects to the reduction of cardiovascular events observed in clinical trials with statins represents one of the major challenges for future studies to understand the antiatherosclerotic benefits of these agents.

Partially modified retro- (PMR) and retro-inverso (PMRI) psi[NHCH(CF(3))]Gly peptides, a conceptually new class of peptidomimetics, have been synthesized in wide structural diversity and variable length by aza-Michael reaction of enantiomerically pure alpha-amino esters and peptides with enantiomerically and geometrically pure N-4,4,4-trifluorocrotonoyl-oxazolidin-2-ones. The factors underlying the observed moderate to good diastereocontrol have been investigated. The conformations of model PMR-psi[NHCH(CF(3))]Gly tripeptides have been studied in solution by (1)H NMR spectroscopy supported by MD calculations, as well as in the solid-state by X-ray diffraction. Remarkable stability of turn-like conformations, comparable to that of parent malonyl-based retropeptides, was evidenced, as a likely consequence of two main factors: 1) severe torsional restrictions about sp(3) bonds in the [CO-CH(2)-CH(CF(3))-NH-CH(R)-CO] module, which is biased by the stereoelectronically demanding CF(3) group and the R side chain; 2) formation of nine-membered intramolecularly hydrogen-bonded rings, which have been clearly detected both in CHCl(3) solution and in some crystal structures. The former factor seems to be more important, as turn-like conformations were found in the solid-state even in the absence of intramolecular hydrogen bonding. The relative configuration of the -C*H(CF(3))NHC*H(R)- stereogenic centers has a major effect on the stability of the turn-like conformation, which seems to require a syn stereochemistry. X-ray diffraction and ab initio computational studies showed that the [-CH(CF(3))NH-] group can be seen as a sort of hybrid between a peptide bond mimic and a proteolytic transition state analogue, as it combines some of the properties of a peptidyl -CONH- group (low NH basicity, CH(CF(3))-NH-CH backbone angle close to 120 degrees, C-CF(3) bond substantially isopolar with the C=O) with some others of the tetrahedral intermediate [-C(OX)(O(-))NH-] involved in the protease-mediated hydrolysis reaction of a peptide bond (high electron density on the CF(3) group, tetrahedral backbone carbon).

We evaluated the expression of two extra-cellular protease systems in a model of spontaneous cerebrovascular pathology: spontaneously hypertensive stroke-prone rats (SHRSP). The appearance of brain damage in individual animals was imaged and followed by means of magnetic resonance imaging (MRI). In situ zymography of brain slices obtained 3 days after the appearance of brain damage showed an increase in plasminogen activator (PA)/plasmin activity that co-localised with the cerebral damage detected by MRI; there was also concomitant accumulation/activation of inflammatory cells in the damaged area. Proteolytic activity was inhibited by the urokinase-specific inhibitor amiloride but not by an antibody against tissue-type plasminogen activator (t-PA). SDS-PAGE zymography of brain extracts revealed the presence of 58 kDa plasminogen-dependent lysis areas in the ischemic and non-ischemic tissues, and a 33 kDa lysis area in ischemic tissue only. An antibody against t-PA inhibited the former, whereas the latter was inhibited by amiloride. The specific induction of urokinase-type plasminogen activator (u-PA) in the damaged tissue was further confirmed by the fact that both u-PA protein mass and mRNA were markedly increased in the damaged cerebral areas. Concomitant metalloproteinase-2 (MMP-2) activation was only observed in the damaged area. These data suggest that u-PA is expressed and selectively catalyses proteolysis in the injured area of spontaneous brain damage in SHRSP.

The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are effective in reducing the risk of coronary events, and are generally very well tolerated. However, simvastatin, lovastatin, cerivastatin and atorvastatin are biotransformed in the liver primarily by cytochrome P450 (CYP) 3A4, and clinical experience has shown that the risk of adverse effect, such as myopathy, increases with concomitant use of statins with drugs that substantially inhibit CYP 3A4 at therapeutic doses. Indeed, pharmacokinetic interactions (e.g. increased bioavailability), myositis, and rhabdomyolysis have been reported following concurrent use of atorvastatin, cerivastatin, simvastatin or lovastatin and cyclosporine A, mibefradil or nefazodone. In contrast, fluvastatin (mainly metabolized by CYP 2C9) and pravastatin (eliminated by other metabolic routes) are less subject to this interaction. Nevertheless, an increase in pravastatin bioavailability has been reported in the presence of cyclosporine A, possibly because of an interaction at the level of biliary excretion. In summary, some statins may have lower adverse drug interaction potential than others, which is an important determinant of safety during long-term therapy.

3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase inhibitors (the so-called statins: atorvastatin, fluvastatin, pravastatin, lovastatin, rosuvastatin and simvastatin) are a well-established class of drugs in the treatment of hypercholesterolemia. Statin monotherapy is generally well tolerated, with a low frequency of adverse events. The most important adverse effects associated with statins are myopathy and an asymptomatic increase in hepatic transaminases, both of which occur infrequently. Since statins are prescribed on a long-term basis, possible interactions with other drugs deserve particular attention, as many patients will typically receive pharmacological therapy for concomitant conditions during the course of statin treatment. Moreover, a combination of therapy between statins and other classes of lipid-lowering agents (e.g., ezetimibe, fibrates, resins and nicotinic acid) is recommended for some patients by current guidelines. Therefore, the potential for drug-drug interactions emerges as a relevant factor in determining the safety profile of statins. This review summarizes the pharmacokinetic properties of statins and emphasizes their clinically relevant drug interactions.

Two key events in atherosclerotic plaque formation are the deposition of lipids in cells of the vascular wall, and migration and proliferation of arterial smooth muscle cells from the tunica intima toward the media. It has been shown that various calcium-channel antagonists may delay plaque formation in animal models. Among these, the new and highly lipophilic calcium antagonists, such as lacidipine and lercanidipine, display the most promising antiatherosclerotic activities. This paper will review and discuss these beneficial effects.

The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on coronary events have generally been attributed to their hypocholesterolaemic properties. However, as mevalonate and other intermediates of cholesterol synthesis (isoprenoids) are necessary for cell proliferation and other important cell functions, effects other than cholesterol reduction may explain the pharmacological properties of statins. In the present review, we discuss the current knowledge on the non-lipid-related effects of statins, with a special emphasis on their potential benefits in different diseases, such as atherosclerosis and cancer. The mechanism(s) responsible for their favourable properties are also reviewed.

Natural AECA constitute a pool of autoantibodies circulating in healthy subjects, which react with a restricted and conserved set of endothelial antigens and establish idiotypic interactions within the immunoglobulin networks. Normal IgG interacts with living endothelial cells and is internalized with a mechanism involving microtubules and resembling that of ligand-receptor internalization. IgG-endothelial cell interaction appears to be dependent on the variable region of antibodies and is followed by modifications of endothelial cell function. Natural AECA increase anti-inflammatory properties of endothelial cells through the selective inhibition of thromboxane A2, endothelin and metalloproteinase-9 secretion, and also through the inhibition of endothelial cell proinflammatory response to TNF-alpha. We have gathered evidence demonstrating that natural AECA constitute a strictly controlled autoantibody pool, interact with living endothelial cells and take part in the regulation of endothelial function, through direct anti-inflammatory effects.

Apolipoprotein E (apoE) mediates the redistribution of lipids among cells and is expressed at highest levels in brain and liver. Human apoE exists in three major isoforms encoded by distinct alleles (ɛ 2 , ɛ 3 , and ɛ 4 ). Compared with APOE ɛ 2 and ɛ 3 , APOE ɛ 4 increases the risk of cognitive impairments, lowers the age of onset of Alzheimer’s disease (AD), and decreases the response to AD treatments. Besides age, inheritance of the APOE ɛ 4 allele is the most important known risk factor for the development of sporadic AD, the most common form of this illness. Although numerous hypotheses have been advanced, it remains unclear how APOE ɛ 4 might affect cognition and increase AD risk. To assess the effects of distinct human apoE isoforms on the brain, we have used the neuron-specific enolase (NSE) promoter to express human apoE3 or apoE4 at similar levels in neurons of transgenic mice lacking endogenous mouse apoE. Compared with NSE-apoE3 mice and wild-type controls, NSE-apoE4 mice showed impairments in learning a water maze task and in vertical exploratory behavior that increased with age and were seen primarily in females. These findings demonstrate that human apoE isoforms have differential effects on brain function in vivo and that the susceptibility to apoE4-induced deficits is critically influenced by age and gender. These results could be pertinent to cognitive impairments observed in human APOE ɛ 4 carriers. NSE-apoE mice and similar models may facilitate the preclinical assessment of treatments for apoE-related cognitive deficits.

The Mediterranean diet, rich in fresh fruits and vegetables, has been associated with a lower incidence of cardiovascular disease and cancer, partly because of its high proportion of bioactive compounds such as vitamins, flavonoids and polyphenols. The major lipid component of such diet is the drupe-derived olive oil, that can be distinguished from other seed oils for the peculiar composition of its non-triglyceride fraction. In fact, several minor components, including polyphenols, grant the oil its particular taste and aroma. Oleuropein, the most abundant among these components, has been shown to be a potent antioxidant endowed with antiinflammatory properties. We investigated the effects of oleuropein on NO release in cell culture and its activity toward nitric oxide synthase (iNOS) expression. The results show that oleuropein dose-dependently enhance nitrite production in LPS-challenged mouse macrophages. This effect was blocked by the iNOS inhibitor L-NAME, indicating increased iNOS activity. Also, Western blot analysis of cell homogenates show that oleuropein increases iNOS expression in such cells. Taken together, our data suggest that, during endotoxin challenge, oleuropein potentiates the macrophage-mediated response, resulting in higher NO production, currently believed to be beneficial for cellular and organismal protection.

Phagocytes play a major role in several diseases. In particular mononuclear phagocyte-derived foam cells have a prominent role in the development of the atherosclerotic lesions. Macrophages are present in all stages of atherogenesis; they internalize lipoproteins and accumulate cholesterol. Moreover, lipid-filled macrophages, by secreting extracellular matrix-degrading enzymes, may weaken rupture-prone atherosclerotic plaques, thus increasing the probability of precipitating atherosclerotic acute symptoms (i.e., myocardial infarction, angina, etc.). Therefore, control of cellular functions and cholesterol accumulation in macrophages represent pharmacological targets against atherosclerosis. In our laboratory we studied the effect of calcium antagonists on cellular cholesterol esterification in cultured macrophages. We also demonstrated that the HMG-CoA reductase inhibitors (vastatins) fluvastatin and simvastatin prevented cholesterol deposition in cultured human and murine macrophage by inhibiting modified LDL endocytosis. Interestingly, vastatin activity was more pronounced in cholesterol-loaded macrophages (i.e., foam cells) than in normal cells. In conclusion, in vitro pharmacological control of cholesterol accumulation in macrophages may be achieved with some calcium antagonists and vastatins independently of their effects on blood pressure or cholesterolemia.

1. Cholesterol esterification and accumulation in the arterial wall is a hallmark of atherogenesis. Several preclinical studies suggest that calcium antagonists may exert antiatherosclerotic activity by directly affecting atherogenesis in the arterial wall. We investigated the effect of the second generation dihydropyridine calcium antagonist lacidipine on cholesterol metabolism in vivo in the aortic arch of cholesterol fed rabbits, and in vitro in mouse cultured peritoneal macrophages. 2. Treatment of cholesterol-fed rabbits with 1, 3 and 10 mg kg-1 day-1 of lacidipine for two months reduced, in a dose-dependent manner, cholesterol esterification in the aortic arch: 24 +/- 6, 30 +/- 12, and 41 +/- 8% inhibition, respectively (P < 0.001 vs HC control). Concomitantly, drug treatment reduced total cholesterol content of the vessel wall. Lacidipine 3 and 10 mg kg-1 day-1 reduced cholesterolaemia (approximately 20%); no effect was observed at the lowest dose used (1 mg kg-1 day-1). These results suggest that the action of lacidipine on cholesterol esterification in the arterial wall involves, at least in part, a direct effect on cellular cholesterol metabolism. Inhibition of cholesterol esterification in the arterial wall was observed also in a reference group of animals treated with the specific ACAT inhibitor CI-976. 3. To evaluate the action of lacidipine on intracellular cholesterol metabolism we performed in vitro experiments with murine macrophages, the main cell type that accumulates cholesterol in the arterial wall. Lacidipine almost completely inhibited cholesterol esterification in cholesterol loaded macrophages in culture. The effect was observed independently of esterification stimuli and in cell free homogenates. The drug modified intracellular cholesterol distribution, doubling the free- to esterified sterol ratio, but did not influence the cellular rate of cholesteryl ester hydrolysis in the cell. All together these results indicate an inhibitory effect of lacidipine on cholesterol esterification catalyzed by the enzyme ACAT in murine macrophages. 4. We concluded that lacidipine influences cellular cholesterol metabolism. This effect may contribute to the potential antiatherosclerotic activity of this drug.

The P2Y(12) receptor is a key player in platelet activation and represents an effective pharmacological target for the inhibition of platelet aggregation and prevention of atherothrombotic events. Indeed, the clinical use of the P2Y(12) receptor inhibitor clopidogrel is an effective strategy for inhibiting platelet activity in patients with acute coronary syndrome, and for preventing thrombotic events in those undergoing percutaneous coronary intervention with stenting. However, clopidogrel has several drawbacks, which include delayed onset of action, large inter-individual variability in platelet response, genetic polymorphism of the metabolizing enzyme, drug-drug interactions (DDIs), and the two-step activation process catalyzed by a series of cytochrome P450 (CYP) isoenzymes. For these reasons, new P2Y(12) receptor inhibitors have been developed in an attempt to improve on the pharmacological and clinical profile of clopidogrel. Three new P2Y(12) receptor inhibitors--prasugrel, cangrelor, and ticagrelor--have arrived, and more are coming into clinical use. Each of these antagonists has individual properties and, according to their mechanism of inhibition, can be divided into irreversible (prasugrel) and reversible inhibitors (ticagrelor, cangrelor). These agents also have different metabolic pathways: prasugrel is a prodrug that requires metabolic activation through a cytochrome-dependent pathway, while ticagrelor and cangrelor do not require metabolic conversion. However, ticagrelor is a CYP3A4 substrate/inhibitor and thus it can be involved in DDIs. Indeed, ticagrelor significantly increases the plasma levels of CYP3A4 substrates such as statins. Moreover, concomitant use with strong CYP3A4 inhibitors (such as ketoconazole, itraconazole, clarithromycin, ritonavir, telithromycin, etc.) is contraindicated, while the co-administration of ticagrelor with potent CYP3A inducers (carbamazepine, rifampicin, phenytoin, phenobarbital) is discouraged. Prasugrel and ticagrelor determine a faster, greater, and more consistent adenosine diphosphate (ADP)-receptor inhibition than clopidogrel, with a near complete inhibition of platelet aggregation between 1-2 h after administration of an oral loading dose, while cangrelor shows a rapid and potent platelet inhibitory effect with intravenous infusion. Thus, the different pharmacokinetic and pharmacodynamic characteristics of the P2Y(12) receptor inhibitors enable clinicians to personalize therapy according to patient-specific medical requirements for better prevention of atherothrombotic events. In the present review, we describe the pharmacological properties, the pharmacokinetic and pharmacodynamic differences, and the clinical efficacy of the currently available P2Y(12) receptor inhibitors.

Previously, we demonstrated in cultured dorsal root ganglion neurons that, in the presence of beta-migrating very low density lipoproteins (beta-VLDL), apolipoprotein (apo) E4, but not apoE3, suppresses neurite outgrowth. In the current studies, murine neuroblastoma cells (Neuro-2a) were stably transfected with human apoE3 or apoE4 cDNA, and the effect on neurite outgrowth was examined. The stably transfected cells secreted nanogram quantities of apoE (44-89 ng/mg of cell protein in 48 h). In the absence of lipoproteins, neurite outgrowth was similar in the apoE3- and apoE4-secreting cells. The apoE4-secreting cells, when incubated with beta-VLDL, VLDL, cerebrospinal fluid lipoproteins (d < 1.21 g/ml), or with triglyceride/phospholipid (2.7:1 (w/w)) emulsions, showed a reduction in the number of neurites/cell, a decrease in neurite branching, and an inhibition of neurite extension, whereas in the apoE3-secreting cells in the presence of a lipid source, neurite extension was increased. Uptake of beta-VLDL occurred to a similar extent in both the apoE3- and apoE4-secreting cells. With low density lipoproteins or with dimyristoylphosphatidylcholine emulsions, either alone or complexed with cholesterol, no differential effect on neurite outgrowth was observed. A slight differential effect was observed with apoE-containing high density lipoproteins. The differential effect of apoE3 and apoE4 in the presence of beta-VLDL was blocked by incubation of the cells with heparinase and chlorate, with lactoferrin, or with receptor-associated protein, all of which prevent the uptake of lipoproteins by the low density lipoprotein receptor-related protein (LRP). The data suggest that the secreted and/or cell surface-bound apoE interact with the lipoproteins and facilitate their internalization via the heparan sulfate proteoglycan-LRP pathway. The mechanism by which apoE3 and apoE4 exert differential effects on neurite outgrowth remains speculative. However, the data suggest that apoE4, which has been shown to be associated with late onset familial and sporadic Alzheimer's disease, may inhibit neuronal remodeling and contribute to the progression of the disease.